This subproject is aimed for continued improvement for safety, efficacy, and in vivo study of lentiviral vectors (LV). We have established a self-inactivating (SIN) LV (pTY), a series of recombinant chimera of murine retrovirus and human immunodeficiency virus type 1 (HIV-1) vectors that contains minimal HIV-1 sequences (less than 500 nt) with deletions of all viral splice sites and the Rev-responsive element (RRE). The SIN vector produced up to 10/7 infectious units per ml of unconcentrated vectors when analyzed by a sensitive Cre/lox recombination assay. Analyses of the SIN LV showed that the 3' polyadenylation processing is compromised due to the large deletion of U3 and U5 sequences. Several strategies will be taken to further increase the efficacy, safety and long term stability of LV. In addition, we have generated novel packing cell lines that employ either the Cre-lox recombination or a Cre-progesterone receptor (PR) fusion system that is inducible by RU486 for a large scale LV production. To study transduction, biodistribution, and host responses in vivo, we will explore a nlCre LV and a sensitive lox/lacZ transgenic mouse model. To achieve these goals, the following four specific aims are proposed: Aim 1: Modifications of the polyadenylation signal of SN LV for improved efficacy and safety, and insertion of insulators for improved long term stability; Aim 2: Analyses of the roles of HIV-RR3 and packing signals in vector function and further characterization of chimeric MLV/HIV-1 vectors; Aim 3: Establishing lentiviral packaging cell lines using the Cre-lox and Cre-PR fusion-RU486 inducible systems; Aim 4: Ex vivo and in vivo LV transduction of lox/lacZ transgenic mice and analyses of host responses. The proposed studies will establish improved LV and obtain necessary preclinical data with the help of the Pathology Core for future clinical LV applications. This subproject will share materials, models, and core facility with the three AAV subprojects, and continue to jointly explore innovative therapeutic models in the program research center.